Efficient chromosomal gene modification with CRISPR/cas9and PCR-based homologous recombination donors in culturedDrosophilacells

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Abstract

The ability to edit the genome is essential for many state-of-the-art experimental paradigms. Since DNA breaks stimulate repair, they can be exploited to target site-specific integration. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9system fromStreptococcus pyogeneshas been harnessed into an efficient and programmable nuclease for eukaryotic cells. We thus combined DNA cleavage bycas9, the generation of homologous recombination donors by polymerase chain reaction (PCR) and transient depletion of the non-homologous end joining factorlig4. Using culturedDrosophila melanogasterS2-cells and the phosphoglycerate kinase gene as a model, we reached targeted integration frequencies of up to 50% in drug-selected cell populations. Homology arms as short as 29 nt appended to the PCR primer resulted in detectable integration, slightly longer extensions are beneficial. We confirmed established rules forS. pyogenes cas9sgRNA design and demonstrate that the complementarity region allows length variation and 5′-extensions. This enables generation of U6-promoter fusion templates by overlap-extension PCR with a standardized protocol. We present a series of PCR template vectors for C-terminal protein tagging and clonalDrosophilaS2 cell lines with stable expression of a myc-taggedcas9protein. The system can be used for epitope tagging or reporter gene knock-ins in an experimental setup that can in principle be fully automated.

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